ECOLOGIA BALKANICA. 2012, Vol. 4, Issue 2 December 2012 pp

ECOLOGIA BALKANICA 2012, Vol. 4, Issue 2 December 2012 pp. 15-23 Study on the Total Coliforms Count and Coli Titter in the Waters of Kardzhali Reservoir, Bulgaria Oliver Y. Todorov, Ivan I. Iliev*, Sonya K. Trifonova University of Plovdiv Paisii Hilendarski, Faculty of Biology, Department of Biochemistry and Microbiology 24, Tsar Asen str., Plovdiv BG-4000, BULGARIA * Corresponding author: e-mail: iziliev@uni-plovdiv.bg Abstract. The aim of this study was to examine TC (total coliforms) and coli titter from two sampling stations in the aquatory of Kardzhali Dam Lake and one station in River Arda, in August, 2011. The values of the TC index in the reservoir vary from 1900±674 cfu/100ml in station I, to 1293±194 cfu/100ml in station II, while the TC value of River Arda reaches 1698±134 cfu/100ml. In Reservoir Kardzhali, the smallest volume of water in which Escherichia coli cells were found, varies between 5 and 15 ml, while for the River Arda the value of coli titter is equal to 1. With highest percentage, regarding the presence of microbiological species in the reservoir waters, is the genus Klebsiella (70%), followed by Citrobacter (15%), Enterobacter (10%) and Serratia (5%), respectively represented by the species Klebsiella oxytoca, Citrobacter freundii, Enterobacter cloacae and Serattia marcescens. In the river Arda two genuses were found - Serratia (50%) and Salmonella (50%). Key words: coli titter, total coliforms, Kardzhali Reservoir. Introduction The permanent control of the sanitary parameters, determining water quality, is used worldwide to monitor and control the quality and safety of various types of water reservoirs, and for prevention of illnesses caused by the polluted water. These infections can arise from the bacteria naturally present in the aquatic environment or microorganisms present in the water as a result of contamination with human or animal faeces (ABRAHAM, 2011). According to a series of authors (ZMYSTLOWSKA et al., 2000а,b; ZMYSTLOWSKA et al., 2003; NIEWOLAK & TUCHOLSKI, 2000a,b), the presence of Escherichia coli in the water is evidence Ecologia Balkanica http://eb.bio.uni-plovdiv.bg for the possible presence of variety of microorganisms, that can cause gastrointestinal diseases (DHSS, 1982; BLACKWOOD, 1978; FAPOHUNDA et al., 1994). Many potential pathogens could be associated with water, so it is thus impractical to screen samples for all possible disease causing agents. Instead various, indicator organisms have been used as surrogate markers of risk. (BARRELL et al., 2000). Most waterborne infectious diseases are related to faecal pollution of water sources therefore aquatic microbiology is mainly based on the need to identify indicators of faecal pollution such as coliforms and E. coli. (HUNTER, 1997). Union of Scientists in Bulgaria Plovdiv University of Plovdiv Publishing House

Study on the Total Coliforms Count and Coli Titter in the Waters of Kardzhali Reservoir, Bulgaria The desirable qualities for a useful water quality indicator are (WHO, 1996): Presence in large numbers in human and warm blooded animal faeces. Easy to identify by simple methods. Does not grow as part from the natural microflora of various types of water reservoirs. Their presence and elimination after treatment of the water is similar to the pathogens. The coliforms are part of the Enterobacteriaceae family, which includes Gram-negative, non-spore-forming, oxidize-negative, straight rods, facultative anaerobic bacteria that ferment lactose with the formation of acid and gas within 24-48h at 35-37 о С. They appear as non-specific indicators of faecal pollution. During the years different schemes are developed for the classification of coliforms. The earliest are the one of MCCONKY (1909) and BERGEY & DEAN (1908). Enough biochemical reactions for the identification of coliforms are established during the 20 th years of the 20 th century (HENDRIKS, 1978). Large numbers of those reactions are still in use today. The main disadvantage is their long duration. In the modern microbiology the efforts are directed in developing reliable, fast methods for detection and determining presence of indicator microorganisms. This directs the efforts towards developing tests for fast identification of coliforms, including basically the genus Escherichia, Klebsiella, Citrobacter, Enterobacter and Serratia. This became possible after the introduction of chromogenic substances, which may be added to the conventional nutrient media. The chromogenic components are modified by the bacterial enzymes or by specific bacterial metabolites; as a result the chromogenic substance changes its colour or fluorescent after the modification. This makes it possible to establish which colonies, possess this specific metabolic activity. It also makes it possible to determine the number of microorganisms from a specific indicator group in a mixed sample. The time required for the determination of different indicator bacteria can be cut down to 16-18h (ASHBOLT, 2001). These methods, although fairly easy, give information primary about the TC represented in the water, but they don t give a clear picture about species affiliation of microorganisms. Often, other species from the family Enterobacteriaceae, which are part of the natural micro flora of the water body, give positive reaction. Therefore further confirmative identification tests are needed. The aim of this study was to determine the total coli forms (TC), coli titter and coli index, by the usage of chromogenic medium, in two sampling stations in the aquatory of Kardzhali dam-lake and in Arda River and to identify the isolated strains. Materials and Methods The research was carried out in August 2011, in the aquatory of Reservoir Kardzhali and Arda River, in the part where the river infuses in the reservoir. The water samples were collected from two sampling stations, located in the different parts of the reservoir: station I - located in close range of the settlement Glavatarci and station II- in the transitional zone of the reservoir (Halach Dere) and one station (III) on the Arda River, in the region where the river flows into the reservoir (Fig. 1). The precise location of the sampling stations is determined with the help of GPS receiver Garmin 76CSx. 16

Oliver Y. Todorov, Ivan I. Iliev, Sonya K. Trifonova Fig. 1. Sampling stations located on Kardzhali Dam Lake and Arda River. The water samples were collected by direct immersion of sterile glass jars with a volume of 500 ml, 50 cm below the water surface and stored in cold conditions until their processing, not later than 6 h after sampling. The coli titter is determined as the smallest volume of water in which the presence of Escherichia coli is established. The coli index is determined as the number of Escherichia coli cells in 1l water sample, and it is calculated based on the coli titter value with the equation: Total coliforms (TC) and E. coli number were determined after filtration of 5 ml water sample through membrane filters (Membrane Solutions) with pore size 0.45 µm, and cultivation on chromogenic medium SLS Coliform Agar (HiChrome) for 48 h on 37 о С. Strains for identification were isolated based on their specific colouring during cultivation on SLS Coliform Agar. From every station 10 strains were isolated. For verification of their purity, strike seeding on Levin medium was used. Biochemical reactions, based on the system for fast identification Enterotube II (ENCISE), were used for identification of the isolated strains. T-test was applied for statistical evaluation of the results (MS Excel). Results and discussion The values for total coliforms, coli titter and coli index from the waters of Kardzhali Reservoir and Arda River are shown in Table 1. Table1. Total coliforms, coli titter and coli index in the waters of Kardzhali Reservoir and Arda River for August, 2011. Parameter Station I II III TC (cfu/ 100 ml) 1900±674 1293±194 1698±134 coli titter (ml/ 1cfu) 5 15 1 coli index (cfu/ 1l) 200 67 1000 The values of TC, established in station II, which is located at the transitional zone of the reservoir between the lake like zone of the water 17

Study on the Total Coliforms Count and Coli Titter in the Waters of Kardzhali Reservoir, Bulgaria body and riverine zone, are within the range of 1293±194 cfu/100ml. They are significantly lower in comparison with the values from station III - 1698±134 cfu/100ml (p<0.05), and station I - 1900±674 cfu/100ml (p<0.05). At the same time, no reliable differences were found between the average values of total coliforms between station I and station III. The long retention time of Kardzhali dam-lake, determines it as a lake like water reservoir, where a continuous process of self-purification takes place (TRAYKOV, 2003). The self-purification of the water is due to the action of physicochemical (continuous water exchange, settling of suspended particles along the longitudinal axis of the reservoir, where microorganisms are adsorbed; bactericidal effect of UV- rays, etc.) and biological (zooplankton; antagonism between the species, etc.) environmental factors. In this kind of water bodies the trophic diversity, and in consequence the quantity of microorganisms, reduces form the tail to the dam of the reservoir (STRASKRABA, 1998). The data deviates from this model. Increased values of the total coliforms in the region of station I, may be due to the influence of village Glavatarci, which is situated in close range of the station, and also from the fact that the study is conducted in a period of intensive tourist season. The settlement does not have two way sewage systems, and this gives the assumption that the sewage waters are filtrated in the reservoir. This assumption is supported by the high values of coli titter, which is an indicator of faecal contamination, from 5 ml water in this station and coli index 200 cfu E. coli/1l. Similar data is shown in the work of other authors, studying the processes of intensive urbanisation in the zones surrounding the large water reservoirs (MALLIN et al., 2000). The biochemical characteristic of the strains isolated for identification and determining the coliform ratio in Kardzhali Reservoir, shows that all strains are Gram-negative, catalase positive, oxidise negative, facultative anaerobic, straight rod-shaped bacteria. When cultivated on Levin medium, they form colonies with purple colouring and metallic shine, which is typical for lactose positive representatives of family Enterobacteriaceae (Bergey`s manual, 2005). Detailed results regarding the biochemical identification of the isolated strains are shown in Tables 2, 3 and 4. The strains isolated from station I are characterized with the highest diversity of species and fall into four genera, based on their biochemical characteristics (Fig. 2a). The Klebsiella genus represents the main share. The strains assimilate glucose with release of acid and gas (CO2), they produce acid from lactose, arabinose and sorbitol, utilize citrate, they have lysine and ornithine activity, tryptophan activity and decompose urea to ammonia. Based on the obtained results all strains of the genus were defined as K. oxytoca (strains 1, 6 and 8) and K. ozaenae (strain 4) (Bergey`s manual 2005, Enterotube II Code Book 2009). Thirty percent (30%) of the studied strains fall into genus Citrobacter. Into this genus fall species that degrade glucose and other carbohydrate sources with the release of acid and gas. The genus is represented by C. freundii (strains 3, 9 and 10), which is normally present in the human microflora. Often it is isolated from clinical patients. 18

Table 2. Biochemical characteristic of the strains isolated from station I of Kardzhali Reservoir Test STRAIN 1 2 3 4 5 6 7 8 9 10 Gram - - - - - - - - - - Oxidase - - - - - - - - - - Catalase + + + + + + + + + + Indol + - + + - + + + - - MR + + + + + + + + + + VP - + - - + - - - - - Citrate + + + + + + + + + + H 2S - - + - - - - - + + Urea + + + - + + + + - - PA - - - - - - - - - - Lysine + - - + - + + + - - Ornithine - + + + + - - - - - Glucose, acid + + + + + + + + + + Glucose, gas + + - - + - - + - - Lactose + + - - + + - + - - L-Arabionse - - + + - - - - + + D-Аdonitol + + + + + + + + + + D-Sorbitol + + + - + + + + + + Dulcitol - - - - - - - - - - O/F F F F F F F F F F F Enterotube II code 34723 32323 33173 26240 32323 24723 24223 34733 21061 21061 In the third place of occurrence is genus Enterobacter, represented by E. cloacae (strains 2 and 5). The species is widely spread in nature as part of the normal microflora of water and soil (Bergey`s manual 2005). They fall into the conditionally pathogenic bacteria in the colon of humans and animals. If they enter elsewhere they can cause urinary tract infections, wound, sepsis etc. (HAIDUSHKA et al., 2011). Strain 7 assimilates all tested carbohydrate sources, with the exception of dulcitol, with the release of acid, without forming gas. It decomposes citrate and decarboxylates amino acids lysine and ornithine in anaerobic conditions, doesn t hydrolyse urea and is indol negative. Based on its biochemical characteristic the strain falls into genus Serratia. All isolated strains from station I fall into the so called faecal coliforms and are used as indicator for faecal pollution (WHO, 1999). Their presence in combination with the high values of coli titre, indicate recent contamination with sewage water of the studied region. 19

Table 3. Biochemical characteristic of the strains isolated from station II of Kardzhali Reservoir Test STRAIN 1 2 3 4 5 6 7 8 9 10 Gram - - - - - - - - - - Оxidase - - - - - - - - - - Catalase + + + + + + + + + + Indol + + + + + + + + + + MR + + + + + + + + + + VP - - - - - - - - - - Citrate + + + + + + + + + + H 2S - - - - - - - - - - Urea + + + + + + + + + + PA - - - - - - - - - - Lysine + + + + + + + + + + Ornithine + + + + + + + + + + Glucose, acid + + + + + + + + + + Glucose, gas + + + + + + + + + + Lactose + + + + + + + + + + L-Arabionse + + + + + + + + + + D-Adonitol + + + + + + + + + + D-Sorbitol + + + + + + + + + + Dulcitol - - - - - - - - - - O/F F F F F F F F F F F Enterotube II code 36763 36763 36763 36763 36763 36763 36763 36763 36763 36763 All isolated strains from station II fall into genus Klebsiella and are represented by the species K.oxytoca (Fig. 2b). Representatives of the species are found in human faeces, but more often are part of the normal microflora of water and soil. Isolated species from station III fall into genus Serratia (strains 1, 2, 3 and 6) and genus Salmonella, respectively represented with the strains 4, 5, 7 and 8 (Fig. 2c). The bacteria of the genus Salmonella are among the main causes of food borne infectious diseases in humans (MOLBAK, 2006). They retain viability up to 4 months in still waters and 5 to 6 days in flowing waters (HAIDUSHKA, 2011). Their high titter in the waters of Arda River is an evidence of contamination with sewage water. This along with the high values of coli titter (1ml) and coli index (200 cfu E. coli/ 1l), creates the risk for occurrence of infectious outbreaks. Conclusions Highest levels of contamination with sewage water, based on the study of coli titter (1ml) and coli index (200 cfu E. coli/ 1l) in the three studied stations, are established in the waters of the Arda River. 20

Table 4. Biochemical characteristic of the strains isolated from station III of Kardzhali Reservoir Test STRAIN 1 2 3 4 5 6 7 8 9 Gram - - - - - - - - - Oxidase - - - - - - - - - Catalase + + + + + + + + + Indol - - - - - - - - + MR + - - + - - - + + VP + + + - + + + - - Citrate + + + + + + + + - H 2S - - - + + - - + - Urea - + - + + - - + - PA - - - - - - - - - Lysine + + + + + + + + + Оrnithine + + + + + + + + + Glucose, acid + + + + + + + + + Glucose, gas + + + + + + + + + Lactose + + + + + + + + + L-Аrabionose + - - + + + - + + D-Аdonitol - - + + - - - - - D-Sorbitol + + + + + + + + + Dulcitol - - - - - - - - - O/F F F F F F F F F F Enterotube II code 36161 36123 36321 37161 37161 36063 35161 37161 36560 a b c Fig 2. Percentage ratio of identified genus form family Enterobacteriaceace by stations (a\station I; b\ station II; c\station III) The high number of coliforms and the species composition is indicative for contamination and loading of Arda River with sewage waters in the section 21

Study on the Total Coliforms Count and Coli Titter in the Waters of Kardzhali Reservoir, Bulgaria before it flows in the Kardzhali Damlake. The absence of pathogenic species, combined with a decrease in the number of TC, coli titter and coli index in station II is evidence of the self-purifying capability of the studied reservoir. The new rise in the number of TC in station I in proximity to Glavatarci village, as well as the presence of faecal coliforms, indicates secondary site of contamination with sewage waters in the region. Acknowledgements The study is funded by the Scientific Research Fund to Ministry of Education, Youth and Science - contract DO 02-307. References ABRAHAM, T., 2011. Food Safety Hazards Related to Emerging Antibiotic Resistant Bacteria in Cultured Freshwater Fishes of Kolkata, India. - Advance Journal of Food Science and Technology 3(1): 69-72. ASHBOLT N., W. GRABOW, M. SNOZZI, 2001. Indicators of microbial water quality. - Water Quality: Guidelines, Standards and Health. BARRELL R., P. HUNTER, G. NICHOLS, 2000. Microbiological standards for water and their relationship to health risk. Commun. Dis. Public Health 2000, 3: 8-13. BRENNER D., N. KRIEG, J. STALEY, 2005. Bergey's Manual of Systematic Bacteriology, Volume II Part C 1414 p. HAIDUSHKA I., M. ATANASOVA, V. KIRINA, Z. KALVACHEV, 2011. Medicine Microbiology, IK-VAP, Plovdiv 352 p. HUNTER P., 1997.Water-borne disease: epidemiology and ecology. Chichester: Wiley. MALLIN, MICHAEL A., KATHLEEN E. WILLIAMS, E. CARTIER ESHAM, R.PATRICK LOWE, 2000. Effect of human development on bacteriological water quality in coastal watersheds. - Ecological Applications, 10:1047 1056. MOLBAK, K., J. OLSEN, H. WEGENER, 2006. Salmonella infections. In: RIEMANN H. AND D. CLIVER (Ed.): Foodborne Infections and Intoxications. Amsterdam, Netherlands. Elsevier Academic Press, pp. 57-136. NIEWOLAK S., S.TUCHOLSKI, 2000b. Ability of bacteriological selfpurification of common carp, tench and crucian carp fingerlings reared in pond receiving the discharge from sewage treatment plant and then kept in running river water of different quality. - Arch. Ryb. Pol.: 8(1): 49-61. NIEWOLAK S., S.TUCHOLSKI, 2000a. Sanitary and bacteriological evaluation of common carp, tench and crucian carp reared in a pond supplied with biologically treated sewage. - Arch. Ryb.Pol. 8(1): 35-48. O HARA C., 2005. Manual and Automated Instrumentation for Identification of Enterobacteriaceae and Other Aerobic Gram-Negative Bacilli. - Clinical Microbiology Reviews, 18(1): 147 162 WHO, 1999. Health-Based Monitoring of Recreational Waters: The Feasibility of a New Approach, Geneva, 50 p. ZMYSLOWSKA I., D. LEWANDOWSKA, E. PIMPICKA, 2000a, Microbial evaluation of water and digestive tract contents of tench (Tinca tinca L.) during tract rearingand. - Arch. Ryb. Pol., 8 (1):95-105. 22

Oliver Y. Todorov, Ivan I. Iliev, Sonya K. Trifonova ZMYSLOWSKA I., I. GOLAS, 2003. Sanitary and bacteriological examination of Lake Oswin water. - Polish Journal of Environmental Studies, 12(3): 351-356. ZMYSLOWSKA I., J. GUZIUR, M. WOZNIAK, M. HARNISZ, C. MIENTKI, 2000b. Effects of culture of the sheatfish (Silurus glanis L.) on the hygienic state of fish and of water environment. - Bulletin Vurh Vodnany, 3: 90-98. Received: 16.07.2012 Accepted: 16.10.2012 Ecologia Balkanica http://eb.bio.uni-plovdiv.bg Union of Scientists in Bulgaria Plovdiv University of Plovdiv Publishing House